Purine receptors are important regulators of intraocular pressure (IOP) since knockout of A3 adenosine receptors lowers mouse IOP, and activation of A1 adenosine receptors lowers IOP in rabbits, mice and monkeys. IOP depends directly on inflow rate and resistance to outflow of aqueous humor, and available data suggest that A3 and A1 receptors likely modulate inflow and outflow, respectively. This proposal integrates membrane transport physiology on a cellular level with measurements at both organ and whole-animal levels to address the fundamental, initiating step in purinergic IOP regulation, release of cellular ATP that also delivers adenosine by ectoenzymatic conversion. Recently, multiple large-bore channels have been found to mediate this release in other preparations. The proposal's major focus is on purinergic regulation of outflow, the site of pathology in glaucoma. Studies of isolated cells will use biochemical, fluorometric, electrophysiologic, pharmacologic and molecular biological measurements. The hypothesis is that: (1) conduits and regulation of ATP release may differ among Schlemm's canal (SC) and trabecular meshwork (TM) cells of the outflow pathway and ciliary epithelial cells of the inflow pathway; (2) identifying the ATP-exit mechanisms by isolated cells, activated by both physiologic and pathophysiologic stimuli, will lead to a strategy for differentially modifying ATP release in the inflow and in different sites of the trabecular outflow pathway; (3) the results will lead to a broader perspective of outflow regulation; and (4) the results may generate new approaches to lower IOP. The major specific aims are: (1) to identify and compare the conduits and regulation of ATP release following physiologic stimulation of SC and TM cells, and to compare these ATP exit pathways with those of ciliary epithelial inflow cells; (2) to exploit relevant pathophysiologic stimuli to activate/inhibit ATP-release channels in normal TM cells and to compare the ATP-release conduits and regulation in TM cells of normal and glaucomatous eyes; and (3) to test whether inhibiting or activating different ATP-release conduits change outflow resistance of isolated, perfused human anterior segments and mouse IOP as predicted from analyses of isolated cells. PUBLIC HEALTH RELEVANCE: Lowering IOP is the only intervention as yet documented to delay the onset and reduce the rate of progression of irreversible blindness from glaucoma. The current proposal seeks to identify the fundamental step in the regulation of IOP by the purine class of signaling molecules. Successful results may lead to an innovative strategy for lowering IOP in glaucoma.